Project Summary/Abstract Knowledge gap: The effects that emanate specifically from mitochondrial DNA damage have been difficult to study because agents that damage mitochondrial DNA also damage nuclear DNA. Because nuclear DNA represents approximately 98% of the DNA in a cell, the demonstrated effects of DNA damage have likely been due to nuclear DNA damage. The simultaneous occurrence of nuclear and mitochondrial DNA damage has resulted in a critical barrier to our understanding of how cells respond specifically to mitochondrial DNA damage. Mouse models have been developed for this project that facilitate experimental regulation of mitochondrial DNA damage in the absence of concurrent nuclear DNA damage. These models will be used to delineate the effects specifically emanating from mitochondrial DNA damage. The overall hypothesis that will be tested is that mitochondrial DNA damage results in mitochondrial, cellular and tissue dysfunction. The specific aims are: 1) to determine the cellular responses to acute mitochondrial DNA damage (such as might occur during trauma) across the mouse lifespan, 2) to determine the effects of chronic mitochondrial DNA damage (such as might occur with diabetes or Parkinson's disease) in the mouse, and 3) to determine if the consequences of mitochondrial DNA damage can be reversed throughout the mouse lifespan. Research design: Transgenic mouse models have been produced that express the restriction endonuclease EcoRI under experimental regulation via the tetracycline system of gene regulation. Regulation of expression can be achieved by supplying the tetracycline analogue, doxycycline, in food to turn off the gene and providing normal chow (no doxycycline) to achieve expression of EcoRI. A mitochondrial translocation presequence fused in frame with the EcoRI coding sequences assures that the protein is translocated to the mitochondrial matrix and not to the nucleus. Once in the mitochondria, EcoRI cleaves the mitochondrial DNA creating damage in the form of double-strand breaks. For aim 1, EcoRI will be turned on at young adult, middle-age or old age to determine how age impacts the effects of acute mitochondrial DNA damage. A number of mitochondrial functions will be examined to determine the effects on mitochondrial function. For aim 2, EcoRI will be turned on in young adults and left on for the lifespan of the mice. Effects on mitochondrial function will be examined at defined timepoints in the lifespan to determine how chronic mitochondrial DNA damage affects mitochondrial function. For aim 3, the ability to reverse the consequences of mitochondrial DNA damage will be examined at defined timepoints in the lifespan. The proposed studies are intended to address the goal of defining effects specifically emanating from mitochondrial DNA damage.